In vitro induction, expression and selection of thioguanine-resistant mutants with human T-lymphocytes.
Conditions have been defined to measure the in vitro induction of mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in human T-lymphocytes by a cell cloning assay. The in vitro growth of mass cultures as well as cell cloning is accomplished by the use of crude T-cell growth factor (TCGF) and irradiated human lymphoblastoid feeder cells. These initial studies employed irradiation of G0 phase peripheral blood mononuclear cells from a single individual. After exposure to gamma-irradiation from a 137Cs source, the cells were stimulated with the mitogen phytohemagglutinin (PHA) and maintained in exponential growth with exogenous TCGF to allow phenotypic expression of the 6-thioguanine-resistant (TGr) mutants. The mutant frequency was determined by measuring cell cloning efficiency in microtiter dishes in the absence and presence of TG, with an optimal selection density of 1 X 10(4) cells/well. The development of this in vitro assay should allow direct study of susceptibility to gamma-irradiation in the human population in terms of both cytotoxicity and mutation induction.
O'Neill
J P
JP
Department of Medicine, University of Vermont, Burlington 05401.
Sullivan
L M
LM
Albertini
R J
RJ
D002478:Cells, Cultured; D002999:Clone Cells; D004351:Drug Resistance / Q000235:genetics / Q000528:radiation effects; D005720:Gamma Rays; D005787:Gene Frequency; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D007376:Interleukin-2 / Q000494:pharmacology; D009152:Mutagenicity Tests; D009154:Mutation; D010835:Phytohemagglutinins / Q000494:pharmacology; D013601:T-Lymphocytes / Q000187:drug effects* / Q000528:radiation effects; D013866:Thioguanine / Q000494:pharmacology*
D007376:Interleukin-2; D010835:Phytohemagglutinins; D007041:Hypoxanthine Phosphoribosyltransferase; D013866:Thioguanine